The present invention relates to complexes of the Neuropilin-1 (NP-1) receptor and a growth factor selected from the group consisting of Vascular Endothelial Growth Factor-B167 (VEGF-B167), Vascular Endothelial Growth Factor-C (VEGF-C), Vascular Endothelial Growth Factor-D (VEGF-D) and the processed form of Vascular Endothelial Growth Factor-B186 (processed VEGF-B186), to methods of using such complexes to induce or antagonize a cellular response mediated by one or more of said growth factors, and to isolated binding partners, such as antibodies, which bind to complexes of one or more of the growth factors with NP-1.
In the developing embryo, the primary vascular network is established by in situ differentiation of mesodermal cells in a process called vasculogenesis. It is believed that all subsequent processes involving the generation of new vessels in the embryo and neovascularization in adults, are governed by the sprouting or splitting of new capillaries from the pre-existing vasculature in a process called angiogenesis (Pepper et al., Enzyme and Protein, 1996 49 138-162; Breier et al., Dev. Dyn. 1995 204 228-239; Risau, Nature, 1997 386 671-674). Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures. In addition to angiogenesis which takes place in the normal individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation, especially of the microvascular system, is increased, such as diabetic retinopathy, psoriasis and arthropathies. Inhibition of angiogenesis is useful in preventing or alleviating these pathological processes.
On the other hand, promotion of angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis, such as in coronary heart disease and thromboangitis obliterans.
The angiogenic process is highly complex and involves the maintenance of the endothelial cells in the cell cycle, degradation of the extracellular matrix, migration and invasion of the surrounding tissue and finally, tube formation. The molecular mechanisms underlying the complex angiogenic processes are far from being understood.
Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis; these include fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGFxcex1), and hepatocyte growth factor (HGF). See for example Folkman et al., J. Biol. Chem., 1992 267 10931-10934 for a review.
It has been suggested that a particular family of endothelial cell-specific growth factors, the vascular endothelial growth factors (VEGFs), and their corresponding receptors is primarily responsible for stimulation of endothelial cell growth and differentiation, and for certain functions of the differentiated cells. These factors are members of the PDGF family, and appear to act primarily via endothelial receptor tyrosine kinases (RTKs).
Nine different proteins have been identified in the PDGF family, namely two PDGFs (A and B), VEGF and six members that are closely related to VEGF. The six members closely related to VEGF are: VEGF-B, described in International Patent Application PCT/US96/02957 (WO 96/26736) and in U.S. Pat. Nos. 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki; VEGF-C, described in Joukov et al., EMBO J., 1996 15 290-298 and Lee et al., Proc. Natl. Acad. Sci. USA, 1996 93 1988-1992; VEGF-D, described in International Patent Application No. PCT/US97/14696 (WO 98/07832), and Achen et al., Proc. Natl. Acad. Sci. USA, 1998 95 548-553; the placenta growth factor (PlGF), described in Maglione et al., Proc. Natl. Acad. Sci. USA, 1991 88 9267-9271; VEGF2, described in International Patent Application No. PCT/US94/05291 (WO 95/24473) by Human Genome Sciences, Inc; and VEGF3, described in International Patent Application No. PCT/US95/07283 (WO 96/39421) by Human Genome Sciences, Inc. Each VEGF family member has between 30% and 45% amino acid sequence identity with VEGF. The VEGF family members share a VEGF homology domain which contains the six cysteine residues which form the cysteine knot motif. Functional characteristics of the VEGF family include varying degrees of mitogenicity for endothelial cells, induction of vascular permeability and angiogenic and lymphangiogenic properties.
Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein that has been isolated from several sources. VEGF shows highly specific mitogenic activity for endothelial cells. VEGF has important regulatory functions in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adult life (Carmeliet et al., Nature, 1996 380 435-439; Ferrara et al., Nature, 1996 380 439-442; reviewed in Ferrara and Davis-Smyth, Endocrine Rev., 1997 18 4-25). The significance of the role played by VEGF has been demonstrated in studies showing that inactivation of a single VEGF allele results in embryonic lethality due to failed development of the vasculature (Carmeliet et al., Nature, 1996 380 435-439; Ferrara et al., Nature, 1996 380 439-442). In addition VEGF has strong chemoattractant activity towards monocytes, can induce the plasminogen activator and the plasminogen activator inhibitor in endothelial cells, and can also induce microvascular permeability. Because of the latter activity, it is sometimes referred to as vascular permeability factor (VPF). The isolation and properties of VEGF have been reviewed; see Ferrara et al., J. Cellular Biochem., 1991 47 211-218 and Connolly, J. Cellular Biochem., 1991 47 219-223. Alterative mRNA splicing of a single VEGF gene gives rise to five isoforms of VEGF.
VEGF-B has similar angiogenic and other properties to those of VEGF, but is distributed and expressed in tissues differently from VEGF. In particular, VEGF-B is very strongly expressed in heart, and only weakly in lung, whereas the reverse is the case for VEGF. This suggests that VEGF and VEGF-B, despite the fact that they are co-expressed in many tissues, may have functional differences. VEGF-B may potentiate the mitogenic activity of low concentrations of VEGF both in vitro and in vivo.
VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique by screening for cellular proteins which might interact with cellular resinoid acid-binding protein type I (CRABP-I). Its isolation and characteristics are described in detail in PCT/US96/02957 and in Olofsson et al., Proc. Natl. Acad. Sci. USA, 1996 93 2576-2581.
VEGF-C was isolated from conditioned media of the PC-3 prostate adenocarcinoma cell line (CRL1435) by screening for ability of the medium to produce tyrosine phosphorylation of the endothelial cell-specific receptor tyrosine kinase VEGFR-3 (Flt4), using cells transfected to express VEGFR-3. VEGF-C was purified using affinity chromatography with recombinant VEGFR-3, and was cloned from a PC-3 cDNA library. Its isolation and characteristics are described in detail in Joukov et al., EMBO J., 1996 15 290-298.
VEGF-D was isolated from a human breast cDNA library, commercially available from Clontech, by screening with an expressed sequence tag obtained from a human cDNA library designated xe2x80x9cSoares Breast 3NbHBstxe2x80x9d as a hybridization probe (Achen et al., Proc. Natl. Acad. Sci. USA, 1998 95 548-553). Its isolation and characteristics are described in detail in International Patent Application No. PCT/US97/14696 (W098/07832).
The VEGF-D gene is broadly expressed in the adult human, but is certainly not ubiquitously expressed. VEGF-D is strongly expressed in heart, lung and skeletal muscle. Intermediate levels of VEGF-D are expressed in spleen, ovary, small intestine and colon, and a lower expression occurs in kidney, pancreas, thymus, prostate and testis. No VEGF-D mRNA was detected in RNA from brain, placenta, liver or peripheral blood leukocytes.
PlGF was isolated from a term placenta cDNA library. Its isolation and characteristics are described in detail in Maglione et al., Proc. Natl. Acad. Sci. USA, 1991 88 9267-9271. Presently its biological function is not well understood.
VEGF2 was isolated from a highly tumorgenic, oestrogen-independent human breast cancer cell line. While this molecule is stated to have about 22% homology to PDGF and 30% homology to VEGF, the method of isolation of the gene encoding VEGF2 is unclear, and no characterization of the biological activity is disclosed.
VEGF3 was isolated from a cDNA library derived from colon tissue. VEGF3 is stated to have about 36% identity and 66% similarity to VEGF. The method of isolation of the gene encoding VEGF3 is unclear and no characterization of the biological activity is disclosed.
Similarity between two proteins is determined by comparing the amino acid sequence and conserved amino acid substitutions of one of the proteins to the sequence of the second protein, whereas identity is determined without including the conserved amino acid substitutions.
VEGF-B exhibits approximately 40 to 45 percent sequence identity to VEGF and approximately 30 percent sequence identity to PlGF. Alternative splicing of PlGF and VEGF-B mRNA""s give rise to two protein isoforms, PlGF-1 and PlGF-2 and VEGF-B167, and VEGF-B186, respectively (Hauser et al., Growth Factors, 9:259 (1993); Maglione et al., Oncogene, 8:925 (1993); Olofsson et al., J. Biol. Chem., 274:19310 (1996)), all of which bind to Flt-1. In both cases, one of these isoforms, PlGF-2 and VEGF-B167, has a basic C-terminus that binds to the cell surface heparin sulfate proteoglycans (HSPGs) and can be released by heparin. The heparin binding region resembles the amino acid sequence encoded by exon 7 of the VEGF gene.
VEGF-C and VEGF-D differ from other members of the VEGF family in that they have N- and C-terminal extensions flanking the VEGF-homology domain and are proteolytically processed (Joukov et al., EMBO J., 16:3898 (1997), Achen et al., Proc. Natl. Acad. Sci. USA, 95:548 (1998)). In addition, VEGF-C is not known to bind heparin, whereas VEGF-B167 and some forms of VEGF (VEGF165, VEGF189 and VEGF206) do. The mature form of VEGF-D lacks the polybasic region that characterizes VEGF/PlGF heparin-binding domains.
PDGF/VEGF family members act primarily by binding to receptor tyrosine kinases. Five endothelial cell-specific receptor tyrosine kinases have been identified, namely VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt4), Tie and Tek/Tie-2. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction. The essential, specific role in vasculogenesis and angiogenesis of VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos.
The only receptor tyrosine kinases known to bind VEGFs are Flt-1/VEGFR-1 [Shibuya et al., Oncogene, 5:519-524 (1990); de Vries et al., Science, 255:989-991 (1992)]; Flk-1/KDR/VEGFR-2 [Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026-30 (1991); Terman et al., Biochem. Biophys. Res. Comm., 187:1579-86 (1992); Millauer et al., Cell,72:835-46 (1993)]; and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with high affinity, and VEGFR-1 also binds VEGF-B and PlGF. VEGF-C has been shown to be the ligand for VEGFR-3, and it also activates VEGFR-2 (Joukov et al., The EMBO Journal, 1996 15 290-298). VEGF-D binds to both VEGFR-2 and VEGFR-3. A ligand for Tek/Tie-2 has been described in International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc. The ligand for Tie has not yet been identified.
VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelial cells. Both VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelia (Oelrichs et al., Oncogene, 1992 8 11-18; Kaipainen et al., J. Exp. Med., 1993 178 2077-2088; Dumont et al., Dev. Dyn., 1995 203 80-92; Fong et al., Dev. Dyn., 1996 207 1-10) and VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., Proc. Natl. Acad. Sci. USA, 1995 9 3566-3570). VEGFR-3 is also expressed in the blood vasculature surrounding tumors.
Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation. Analysis of embryos carrying a completely inactivated VEGFR-1 gene suggests that this receptor is required for functional organization of the endothelium (Fong et al., Nature, 1995 376 66-70). However, deletion of the intracellular tyrosine kinase domain of VEGFR-1 generates viable mice with a normal vasculature (Hiratsuka et al., Proc. Natl. Acad. Sci. USA 1998 95 9349-9354). The reasons underlying these differences remain to be explained but suggest that receptor signalling via the tyrosine kinase is not required for the proper function of VEGFR-1. Analysis of homozygous mice with inactivated alleles of VEGFR-2 suggests that this receptor is required for endothelial cell proliferation, hematopoesis and vasculogenesis (Shalaby et al., Nature, 1995 376 62-66; Shalaby et al., Cell, 1997 89 981-990). Inactivation of VEGFR-3 results in cardiovascular failure due to abnormal organization of the large vessels (Dumont et al. Science, 1998 282 946-949).
Although VEGFR-1 is mainly expressed in endothelial cells during development, it can also be found in hematopoetic precursor cells during early stages of embryogenesis (Fong et al., Nature, 1995 376 66-70). In adults, monocytes and macrophages also express this receptor (Barleon et al., Blood, 1996 87 3336-3343). In embryos, VEGFR-1 is expressed by most, if not all, vessels (Breier et al., Dev. Dyn., 1995 204 228-239; Fong et al., Dev. Dyn., 1996 207 1-10).
The receptor VEGFR-3 is widely expressed on endothelial cells during early embryonic development but as embryogenesis proceeds becomes restricted to venous endothelium and then to the lymphatic endothelium (Kaipainen et al., Cancer Res., 1994 54 6571-6577; Kaipainen et al., Proc. Natl. Acad. Sci. USA, 1995 92 3566-3570). VEGFR-3 is expressed on lymphatic endothelial cells in adult tissues. This receptor is essential for vascular development during embryogenesis. Targeted inactivation of both copies of the VEGFR-3 gene in mice resulted in defective blood vessel formation characterized by abnormally organized large vessels with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at post-coital day 9.5. On the basis of these findings it has been proposed that VEGFR-3 is required for the maturation of primary vascular networks into larger blood vessels. However, the role of VEGFR-3 in the development of the lymphatic vasculature could not be studied in these mice because the embryos died before the lymphatic system emerged. Nevertheless it is assumed that VEGFR-3 plays a role in development of the lymphatic vasculature and lymphangiogenesis given its specific expression in lymphatic endothelial cells during embryogenesis and adult life. This is supported by the finding that ectopic expression of VEGF-C, a ligand for VEGFR-3, in the skin of transgenic mice, resulted in lymphatic endothelial cell proliferation and vessel enlargement in the dermis. Furthermore this suggests that VEGF-C may have a primary function in lymphatic endothelium, and a secondary function in angiogenesis and permeability regulation which is shared with VEGF (Joukov et al., EMBO J., 1996 15 290-298).
Some inhibitors of the VEGF/VEGF-receptor system have been shown to prevent tumor growth via an anti-angiogenic mechanism; see Kim et al., Nature, 1993 362 841-844 and Saleh et al., Cancer Res., 1996 56 393-401.
Recently, a novel 130-135 kDa VEGF isoform specific receptor has been purified and cloned (Soker et al., Cell, 1998 92 735-745). The VEGF receptor was found to specifically bind the VEGF165 isoform via the exon 7 encoded sequence, which shows weak affinity for heparin (Soker et al., Cell, 1998 92 735-745). Surprisingly, the receptor was shown to be identical to human neuropilin-1 (NP-1), a receptor involved in early stage neuromorphogenesis. PlGF-2 also appears to interact with NP-1 (Migdal et al., J. Biol. Chem., 1998 273 22272-22278). NP-1 expression enhanced the binding of VEGF165 to KDR and enhanced the bioactivity of VEGF156 (Soker et al., Cell, 92:735-745 (1998)).
NP-1 has been identified as a receptor that mediates chemorepulsant activity of the collapsin/semiphorins (collapsin-1/Sema III/Sema D), a large family of transmembrane and secreted glycoproteins that function in repulsive cone and axon guidance [See Kolodkin et al., Cell, 75:1389-1399 (1993); Zhigang et al., Cell, 90:739-751 (1997); Kolodkin et al., Cell, 90: 753-762 (1997)]. The Kd of Sema III binding to NP-1, 2xe2x88x923xc3x9710xe2x88x9210M, is similar to the Kd of VEGF165 to NP-1, 2xe2x88x923xc3x9710xe2x88x9210M (Zhigang et al., Cell, 90:739-751 (1997); Kolodkin et al., Cell, 90: 753-761; Soker et al., Cell 92:735745(1998)). These findings are especially surprising because two structurally different ligands with markedly different biological activities (namely, stimulation of angiogenesis by VEGF and chemorepulsion of neuronal cells by Sema III) bind to the same receptor and with similar affinity.
NP-1 expression is found in some tumor cell lines, and is prominent in some organs where VEGF-B is highly expressed, such as heart, pancreas and skeletal muscle.
The present invention is based on the discovery that VEGF-B167, VEGF-C, VEGF-D and processed VEGF-B186 are each capable of binding to the extracellular domain of NP-1 to form bioactive complexes which mediate useful cell responses and/or antagonize undesired biological activities.
According to the present invention, it has been found that VEGF-B167 binds to the NP-1 receptor via the exon 6B encoded sequence in the region responsible for heparin binding. Binding also occurs between NP-1 and PlGF-2, VEGF-C, VEGF-D and processed VEGF-B186. 
VEGF-B exists in various isoforms as a result of alternative splicing. VEGF-B167 is composed of exons 1 through 5 plus exon 6B and exon 7, with the stop codon being in exon 7. VEGF-B186 is composed of exons 1 through 5 plus exon 6A, and it terminates in exon 6B, albeit in a different reading frame than that used in exon 6B for VEGF-B167. Exon 7 is not translated in VEGF-B186. As noted, VEGF-B167 binds to NP-1. The fact that the product of a construct expressing only exons 1 through 5 (murine VEGF-BkEX1xe2x88x925 as described in PCT/US97/23533, which is incorporated herein by reference) does not bind NP-1 indicates that the binding site is not in any of exons 1 through 5. Similarly, the fact that processed VEGF-B186 does not contain the expression product of exon 7 but can still bind NP-1 indicates that the VEGF-B exon 6B encoded region of VEGF-B167 binds NP-1.
The processed form of VEGF-B186 also can bind NP-1 and yet does not contain exon 6B. Although the location of the cleavage site which creates the processed form of VEGF-B186, as opposed to the full length form, is not known, the processed form comprises exons 1-5 and at least part of exon 6A. Thus, there may be a second binding site coded within the part of exon 6A which is expressed in processed VEGF-B186. 
According to the present invention, it has been found that VEGF-B, VEGF-C, VEGF-D and other ligands are modular in that the domains may be expressed separately from each other and disassociated. On the cell surface, with respect to VEGF-B167, one domain binds Flt-1 and another domain binds neuropilin and heparin sulfate. This independent modularity and dissassociability may be exploited for example, by joining co-receptor binding domains.
Also, it has been found that VEGF-C binds with VEGFR-2 and VEGFR-3 and neuropilin receptors. VEGF-Cxcex94Nxcex94C does not bind NP-1 while full length VEFG-C does.
References herein to the amino acid sequence of VEGF-B refer to the sequence for human VEGF-B described in Eriksson et al., published PCT Application No. WO 96/26736 (Genbank database accession no. U52819). References to the amino acid or nucleotide sequences of NP-1 refer to the sequences described by Soker et al., Cell, 92:735-745 (1998), (EMBL database accession no. AF016050). A useful method for assaying endothelial cell proliferation is described in Olofsson et al., Proc. Natl. Acad. Sci. USA, 93:2576-81 (1996).
A sample containing the receptor protein could be, for example, soluble NP-1 receptor produced naturally in the conditioned medium of cells that normally express the receptor. Tissue samples or tissue fluids shed naturally from cells by proteolytic events also could be used as receptor samples.
An analog or functional analog refers to a modified form of the respective polypeptide in which at least one amino acid substitution has been made such that the analog retains substantially the same biological activity as the unmodified polypeptide in vivo and/or in vitro. In addition, analogs of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 identified by this aspect of the invention may be small molecules, for example proteins or peptides or non-proteinaceous compounds. The analogs also could include VEGF-B167, VEGF-C, VEGF-D, processed VEGF-B186 or derivatives thereof (including, but not limited to, fragments of monomers or dimers) tagged with a toxin or drug or radioactive isotope which could target NP-1 expressed and upregulated on endothelial cells in tumors. Such molecules could be useful to antagonize or inhibit unwanted cellular responses induced by members of the VEGF family of growth factors, such as tumor-induced angiogenesis or psoriasis or retinopathies by techniques analogous to those described in Kim et al., Nature, 362:841-44 (1993) or Aiello et al., New England Journal of Medicine, 331:1480-87 (1994).
Abolishing interaction of NP-1 with VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 could alleviate biological activity in cancer. It is also possible that VEGF-B167 signals without Flt-1 but via NP-1 by an as yet unknown mechanism in cells. Such signaling may exist in normal and tumor cells which express NP-1. Treatments that inhibit this signaling may be useful in treating cancer and/or in modulation of normal or diseased vasculature.
One procedure for isolating complexes of NP-1 with VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 involves using fusion proteins of the NP-1 receptor, preferably the extracellular domain of NP-1, and immunoglobulin G (IgG) followed by Sepharose A binding. Alternatives to the use of Sepharose A include using ion-exchange chromatography, gel filtration or affinity chromatography. Conditioned medium containing receptor/IgG fusion proteins could be allowed to interact with conditioned medium either from cells either transfected with DNA encoding the respective growth factor ligand or analog thereof, or from cells which naturally express the ligand, or with a solution containing a candidate ligand analog.
Conditioned medium from cultures of cells expressing NP-1-IgG fusion proteins can be passed over a Sepharose A column or matrix to immobilize the receptor, or the fusion proteins can be immobilized in cellulose disks or absorbed onto plastic in the form of an ELISA test. A second solution containing conditioned medium from cells expressing the ligand is then passed over immobilized receptor. If desired, the ligand may be radioactively labeled in order to facilitate measurement of the amount of bound ligand by radioassay techniques.
Such an assay can be used to screen for conditions involving overexpression of the NP-1 receptor, i.e. through detection of increased bound radioactivity compared to a control. This methodology can also be used to screen for the presence of analogs which compete with VEGF-B167, VEGF-C, VEGF-D and/or processed VEGF-B186, i.e. through detection of decreased bound radioactivity compared to a control indicative of competition between the radioactively labeled ligand used in the test and a non-radioactive putative analog.
Examples of detectable cellular responses to members of the VEGF family include endothelial cell proliferation, angiogenesis, tyrosine phosphorylation of receptors, and cell migration. Isoform specific induction may be accomplished. Specific examples include urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1(PAI-1) induction, endothelial cell migration and growth stimulation of some endothelial cell types. Tube formation is also possible.
The cells which express the receptor protein or a portion thereof may be cells which naturally express the receptor, or they may be cells into which the nucleic acid encoding the receptor or a portion thereof is introduced such that the receptor protein or a portion thereof is expressed. Transfection or transduction of nucleic acid encoding neuropilin could be performed to increase expression in cells in vitro and in vivo. Expression in cells not expressing neuropilin, [e.g. hematopoietic cells, see Soker et al., Cell, 92:735-745 (1998)] should make them more responsive to induction. Induction should occur by VEGF-B167, VEGF-C, VEGF-D or the processed form of VEGF-B186, but induction should not occur by the nonprocessed (i.e., full length) form of VEGF-B186. Also, expression of NP-1 or a portion thereof by cells adjacent to those expressing a particular RTK could also modulate the effective concentrations of the RTK and/or signaling by the RTK.
Wherever the introduction of a nucleotide sequence encoding NP-1, an extracellular domain thereof, a ligand-binding fragment or analog thereof, or a polypeptide chain having binding affinity for VEGF-B167 or VEGF-C and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the ligand-binding fragment is referred to herein, this may be effected by transformation with a plasmid vector, or transfection either ex vivo or in vivo with a viral vector (e.g., adenoviruses, pox viruses or retroviruses),or with bacterial vectors (e.g., BCG) or by injection of naked DNA.
Heparin may modulate NP-1 binding by those growth factors which bind heparin, and a basic heparin binding peptide could modulate it independent of direct NP-1 interaction. VEGF-C does not bind heparin, so VEGF-C would not be subject to heparin modulation.
Alternatively, the foregoing assay could be reversed by immobilizing the VEGF-B167 ligand or candidate analog and contacting the immobilized ligand with conditioned medium from cells expressing the receptor or a portion of the receptor, such as the extracellular domain or the ligand-binding fragment.
Means for detecting protein/receptor binding may comprise, for example, means for detecting specific binding interaction of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 or an analog of any of them with the NP-1 receptor protein or portion thereof or means for detecting induction of a cellular response induced by VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186, such as uPA and PAI-1 induction, and receptor phosphorylation.
A still further aspect of the invention relates to an isolated ligand-receptor complex comprising two molecules, one defining the ligand and comprising the NP-1 binding sequence of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 (for example, the exon 6B encoded sequence of VEGF-B167) or a receptor-binding analog thereof, and the second defining the receptor and being selected from the group consisting of a polypeptide chain comprising an amino acid sequence of NP-1, the extracellular domain thereof, the ligand-binding domain, a receptor analog of NP-1 which binds VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 and which has at least 90% amino acid identity with the ligand-binding domain, and a polypeptide chain having binding affinity for VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the ligand-binding fragment. Preferably the receptor is the NP-1 receptor, and the ligand includes the NP-1-binding sequence of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 (for example, the exon 6B encoded NP-1-binding sequence of VEGF-B167).
Isolation and purification of the ligands or complexes could be effected by conventional procedures such as immunoaffinity purification using monoclonal antibodies according to techniques described in standard reference works such as Harlow et al., Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press (1988) and/or Marshak et al., Strategies for Protein Purification and Characterization, Cold Spring Harbor Laboratory Press (1996). Suitable antibodies to the individual ligands or to the complexes could be generated by conventional techniques.
A cell-free complex could be used either in vivo or in vitro to compete with binding of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 to a receptor or to prevent dimerization of the cell-bound receptor after ligand binding. Such a cell-free complex would comprise at least one receptor molecule, for example soluble NP-1 (sNP-1), and a dimer molecule. The sNP-1 is defined as a non-membrane bound protein as well as a portion of the receptor, such as the extracellular domain or the ligand-binding fragment of NP-1. The dimer molecule may be a homodimer or mixed dimer of VEGF-B176, VEGF-C, VEGF-D or processed VEGF-B186 or an analog of any of them. One molecule of the dimer can be bound to the receptor molecule in the complex and the second molecule of the dimer has a free binding site available to bind to a cell surface receptor.
It is also an aspect of the present invention to provide an isolated binding partner having specific binding affinity for an epitope on a ligand-receptor complex comprising VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 protein or an analog thereof in specific binding interaction with the ligand binding domain of a receptor having a polypeptide chain comprising an amino acid sequence of NP-1, the extracellular domain or the ligand-binding domain of NP-1, a receptor analog thereof which binds VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 and which has at least 90% amino acid identity with the ligand-binding domain of NP-1, or a polypeptide chain having binding affinity for VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the ligand-binding domain of NP-1; wherein the binding partner has substantially no binding affinity for uncomplexed VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 or analog thereof. Exemplary stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in 5xc3x97SSC, 20 mM NaPO4, pH 6.8, 50% formamide; and washing at 42xc2x0 C. in 0.2xc3x97SSC. Those skilled in the art understand that it is desirable to vary these conditions empirically based on the length and the GC nucleotide base content of the sequences to be hybridized, and that formulas for determining such variation exist. See for example Sambrook et al, xe2x80x9cMolecular Cloning: A Laboratory Manualxe2x80x9d, Second Edition, pages 9.47-9.51, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1989). Preferably the binding partner also will have substantially no binding affinity for any uncomplexed form of the receptor protein or receptor analog thereof. The binding partner may be an antibody which reacts with or recognizes such growth factor/receptor complexes. Either polyclonal or monoclonal antibodies may be used, but monoclonal antibodies are preferred. Such antibodies can be made by standard techniques, screening out those that bind to either receptor or ligand individually.
As used in this application, percent sequence identity is determined by using the alignment tool of xe2x80x9cMEGALIGNxe2x80x9d from the Lasergene package (DNASTAR, Ltd. Abacus House, Manor Road, West Ealing, London W130AS United Kingdom) and using its preset conditions. The alignment is then refined manually, and the number of identities are estimated in the regions available for a comparison.
An additional aspect of the invention relates to the use of an antagonist to VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 obtained according to the methods described above for
(i) antagonizing binding of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 to a cell surface receptor, or
(ii) antagonizing induction of a cellular response mediated by VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186.
A preferred antagonist comprises an antibody having binding specificity for
(i) the ligand binding domain of a protein having a polypeptide chain comprising an amino acid sequence of NP-1, an analog thereof which binds VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 and which has at least 90% amino acid identity with the ligand-binding domain of NP-1, or a polypeptide chain having binding affinity for VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the ligand-binding domain of NP-1; or
(ii) a receptor binding domain having a binding affinity for the NP-1 receptor and comprising the NP-1-binding sequence of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 (for example, the exon 6B encoded NP-1-binding sequence of VEGF-B167).
The ligand binding domain of a protein having binding affinity for VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 desirably will exhibit at least 90%, preferably at least 95%, amino acid identity with the ligand-binding domain of NP-1 and especially preferably will correspond thereto. The receptor binding domain of a VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 analog desirably will exhibit at least 90%, preferably at least 95%, sequence identity with the NP-1-binding sequence of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 (e.g., with the exon 6B encoded NP-1-binding sequence of VEGF-B167), and especially preferably will correspond thereto.
Yet another aspect of the invention relates to the use of a protein selected from the group consisting of a polypeptide chain comprising an amino acid sequence of NP-1, the extracellular domain, the ligand-binding domain of NP-1, or an analog thereof which binds VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 and which has at least 90% amino acid identity with the ligand-binding domain of NP-1, and a polypeptide chain having binding affinity for VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the ligand-binding domain of NP-1; in a method for antagonizing:
(a) binding of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 to a protein, e.g. a cell surface receptor, or
(b) induction of a cellular response mediated by VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186. 
The polypeptide chain competes with the cell surface receptor for the respective growth factor and ties up the available growth factor, thereby preventing it from effectively interacting with the cell surface receptor and inducing the cellular response normally mediated by that growth factor. A suitable peptide chain could be a solubilized form of the receptor (sNP-1) as described in Soker et al., Cell, 92:735-745 (1998).
Additionally, it is an aspect of the invention to provide a method for antagonizing binding of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 to a cell surface receptor, the method comprising the step of providing a protein having binding specificity for the amino acid sequence of NP-1 or a sequence variant thereof which binds a receptor for VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186, wherein the protein has at least 90%, and preferably at least 95%, amino acid sequence identity with the NP-1-binding sequence of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186, such that the protein, when provided to a cell expressing the cell surface receptor, is competent to interact specifically with the receptor and thereby substantially inhibits binding of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186, respectively, to the receptor. The protein may desirably be an analog of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 obtained according to one of the methods described above, preferably an analog which does not induce cellular activities mediated by VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186. 
The interacting domain in neuropilin or an isolated soluble NP binding domain or peptide of VEGF-B167, VEGF165, PlGF-2, VEGF-C or VEGF-D could be used as a soluble inhibitor of the full effects of any VEGF-B167, VEGF165, PlGF-2, VEGF-C or VEGF-D. The VEGF-B167, VEGF165, PlGF-2, VEGF-C or VEGF-D derived domain or peptide could also be anchored to the cell surface by various means, such as expression vector transformation, or linked domains with affinity to other cell surface molecules.
In accordance with a further aspect of the invention, pharmaceutical preparations are provided which comprise such growth factor/receptor complexes.
In yet another aspect of the invention a method is provided for treating a disease state characterized by overexpression of an NP-1 cell surface receptor, said method comprising administering to a patient suffering from said disease state an effective NP-1 receptor binding or receptor antagonizing amount of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 or an analog thereof obtained according to one of the methods described above.
NP-1 knockout and overexpression studies have shown interesting effects in the cardiovascular system, especially in the heart, where VEGF-B167 is probably important. VEGF-B167, VEGF-C, VEGF-D, processed VEGF-B186 or derivatives thereof could also modulate the binding of the other ligands of NP-1, namely collapsin-1/semaphorinIII/D, semaphorin E and semaphorinIV, and in this way VEGF-B167, VEGF-C, VEGF-D, processed VEGF-B186 and/or derivatives thereof may affect the central nervous system.
Where the receptor protein comprises a polypeptide chain other than residues of NP-1 but which nevertheless exhibits a binding affinity for VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186, it should exhibit at least 80%, desirably at least 85%, preferably at least 90%, and especially preferably at least 95%, amino acid identity with the ligand-binding domain of NP-1. Similarly, useful analogs of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186 should exhibit, respectively, at least 80%, preferably at least 85%, particularly preferably at least 90%, and especially preferably at least 95%, sequence identity to NP-1 binding domain of VEGF-B167, VEGF-C, VEGF-D or processed VEGF-B186.